Fluorimetric detection of pathogenic bacteria using magnetic carbon dots

A novel and facile approach of pathogenic bacteria detection, which utilizes fluorescent sensing and bacteria capture with Magnetic carbon dots (Mag-CDs), was proposed in this work. Magnetic nanoparticles were synthesized and then decorated with C-dots, and further functionalized with amine groups (chitosan). In this way, bacteria were strongly anchored on the hybrid material Mag-CDs for highly sensitive fluorescent detection. The Mag-CDs were characterized by UV–vis, FT-IR spectra, TEM images, XRD, and EDX. The characterizations validate the fabrication of amine-Mag-CDs and the promising applications of this material. Fluorescence spectroscope and MALDI-MS were used for the detection and identification of bacterial strains, respectively. The limit of detection for Staphylococcus aureus and Escherichia coli was found to be 3 × 10² and 3.5 × 10² cfu mL⁻¹, respectively. With these encouraging results, it is expected that it would open revenues for promising applications of Mag-CDs nanomaterial.     

柠檬酸盐稳定的水溶性CdSe量子点的合成及表征

以柠檬酸三钠为稳定剂在水溶液中合成了水溶性CdSe量子点,用X射线粉末衍射、透射电镜、紫外-可见吸收光谱和荧光发射光谱对CdSe量子点的结构、形貌及其荧光性质进行了表征.结果表明合成的CdSe量子点为立方闪锌矿结构,呈球形,分散性良好,平均尺寸约为2.6nm,具有窄且对称的荧光发射光谱,半峰宽为45nm.     

Calorimetric investigation of the effect of hydroxyanthraquinones in Rheum officinale Baill on Staphylococcus aureus growth

The inhibitory effects of five hydroxyanthraquinones (HAQs) from root and rhizoma of Rheum officinale Baill, a traditional Chinese medicinal (TCM) herb, on Staphylococcus aureus growth were investigated by calorimetry. The power-time curves of S.aureus with and without HAQ were acquired and the extent and duration of inhibitory effects on the metabolism evaluated by growth rate constants (k₁, k₂), half inhibitory ratio (IC₅₀), maximum heat output (P_max) and peak time (t_p). The value of k₁ and k₂ of S. aureus in the presence of the five HAQs decreased with the increasing concentrations of HAQs. Moreover, P_max was reduced and the value of t_p increased with increasing concentrations of the five drugs. The inhibitory activity varied for different drugs. IC₅₀ of the five HAQs was 4 μg ml⁻¹ for emodin, 3.5 μg ml⁻¹ for rhein, 10 μg ml⁻¹ for aloe-emodin, 1000 μg ml⁻¹ for chrysophanol, 1600 μg ml⁻¹ for physcion. The sequence of antimicrobial activity of the five HAQs: rhein > emodin > aloe-emodin > chrysophanol > physicion.     

Dual-color upconversion fluorescence and aptamer-functionalized magnetic nanoparticles-based bioassay for the simultaneous detection of Salmonella Typhimurium and Staphylococcus aureus

A sensitive luminescent bioassay for the simultaneous detection of Salmonella Typhimurium and Staphylococcus aureus was developed using aptamer-conjugated magnetic nanoparticles (MNPs) for both recognition and concentration elements and using upconversion nanoparticles (UCNPs) as highly sensitive dual-color labels. The bioassay system was fabricated by immobilizing aptamer 1 and aptamer 2 onto the surface of MNPs, which were employed to capture and concentrate S. Typhimurium and S. aureus. NaY_0.78F₄:Yb_0.2,Tm_0.02 UCNPs modified aptamer 1 and NaY_0.28F₄:Yb_0.70,Er_0.02 UCNPs modified aptamer 2 further were bond onto the captured bacteria surface to form sandwich-type complexes. Under optimal conditions, the correlation between the concentration of S. Typhimurium and the luminescent signal was found to be linear within the range of 10¹–10⁵ cfu mL⁻¹ (R² = 0.9964), and the signal was in the range of 10¹–10⁵ cfu mL⁻¹ (R² = 0.9936) for S. aureus. The limits of detection of the developed method were found to be 5 and 8 cfu mL⁻¹ for S. Typhimurium and S. aureus, respectively. The ability of the bioassay to detect S. Typhimurium and S. aureus in real water samples was also investigated, and the results were compared to the experimental results from the plate-counting methods. Improved by the magnetic separation and concentration effect of MNPs, the high sensitivity of UCNPs, and the different emission lines of Yb/Er- and Yb/Tm-doped NaYF₄ UCNPs excited by a 980 nm laser, the present method performs with both high sensitivity and selectivity for the two different types of bacteria.     

A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots

Immunoassay is a powerful tool for rapid detection of food borne pathogens in food safety monitoring. However, conventional immunoassay always suffers from low sensitivity when it employs enzyme-catalyzing chromogenic substrates to generate colored molecules as signal outputs. In the present study, we report a novel fluorescence immunoassay for the sensitive detection of E. coli O157:H7 through combination of the ultrahigh bioactivity of catalase to hydrogen peroxide (H₂O₂) and H₂O₂-sensitive mercaptopropionic acid modified CdTe QDs (MPA-QDs) as a signal transduction. Various parameters, including the concentrations of anti-E. coli O157:H7 polyclonal antibody and biotinylated monoclonal antibody, the amounts of H₂O₂ and streptavidin labeled catalase (CAT), the hydrolysis temperature and time of CAT to H₂O₂, as well as the incubation time between H₂O₂ and MPA-QDs, were systematically investigated and optimized. With optimal conditions, the catalase-mediated fluorescence quenching immunoassay exhibits an excellent sensitivity for E. coli O157:H7 with a detection limit of 5 × 10² CFU/mL, which was approximately 140 times lower than that of horseradish peroxidase-based colorimetric immunoassay. The reliability of the proposed method was further evaluated using E. coli O157:H7 spiked milk samples. The average recoveries of E. coli O157:H7 concentrations from 1.18 × 10³ CFU/mL to 1.18 × 10⁶ CFU/mL were in the range of 65.88%–105.6%. In brief, the proposed immunoassay offers a great potential for rapid and sensitive detection of other pathogens in food quality control.     

Sensitive and simple detection of Escherichia coli strain based on time-resolved fluorescence DNA hybridization assay

A two-probe tandem DNA hybridization assay based on time-resolved fluorescence was employed to detect Escherichia coli strain. The amino modified capture probe was covalently immobilized on the common glass slide surface. The Eu(TTA)₃(5-NH₂-phen) with the characteristics of long lifetime and intense luminescence was labeled with reporter probe. The original extracted DNA samples without the purification and amplification process were directly used in the hybridization assay. The concentration of capture probe, hybridization temperature, hybridization and washing time were optimized. The detection limit is about 1.49 × 10³ CFU mL⁻¹E. coli cells, which is comparable to the value of most microbiology methods. The proposed method has the advantages of easy operation, satisfactory sensitivity and specificity, which can provide a promising technique for monitoring the microorganisms.     

Determination of methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteria in blood by capillary zone electrophoresis

Serious bloodstream infections are a significant complication in critically ill patients. The treatment of these infections has become more difficult because of the increasing prevalence of multiresistant strains, especially methicillin-resistant Staphylococcus aureus (MRSA). Rapid differentiation of low number of MRSA from methicillin-susceptible S. aureus (MSSA) cells (10¹–10² cells mL⁻¹) in blood is necessary for fast effective antibiotic therapy. Currently, three groups of techniques, phenotyping, genotyping, and mass spectrometry, are used for MRSA and MSSA strains differentiation. Most of these techniques are time-consuming. PCR and other molecular techniques allow the detection and differentiation between MSSA and MRSA directly from blood cultures. These methods alone are rapid and they have good reproducibility and repeatability. Potential disadvantages of the genotyping methods include their discrimination ability, technical complexity, financial costs, and difficult interpretation of the results.     

An immunochemical strategy based on peptidoglycan synthetic peptide epitopes to diagnose Staphylococcus aureus infections

The characteristic pentaglycyl cross-bridge of the Staphylococcus aureus peptidoglycan (PG) cell wall component is an attractive epitope to raise specific antibodies against this microorganism. Based on this approach, we report here for the first time a competitive ELISA able to detect S. aureus down to 10⁴ CFU mL⁻¹, without pre-enrichment on cell culture. The antibodies were raised against peptide-protein bioconjugates prepared by covalently coupling peptide haptens (PSau6 and PSau8) designed and synthesized taking into consideration the complex tridimensional structure in the PG polymer. Deglycosylation of the PG under acidic conditions has found to increase assay detectability. Assay performance has been evaluated in clinical samples such as bronchoalveolar lavage (BAL) and bronchoalveolar endotracheal aspirates (BAS) showing promising results for further implementation of this immunoassay as a daily routine diagnostic tool. Cross-reactivity studies have demonstrated that the immunoassay is specific for S. aureus.     

Combining biofunctional magnetic nanoparticles and ATP bioluminescence for rapid detection of Escherichia coli

A rapid, specific and sensitive method for assay of Escherichia coli (E. coli) using biofunctional magnetic nanoparticles (BMNPs) in combination with adenosine triphosphate (ATP) bioluminescence was proposed. The BMNPs were fabricated by immobilizing a specific anti-E. coli antibody on the surface of amine-functionalized magnetic nanoparticles (about 20 nm in diameter), and then was applied to capture the target bacteria E. coli from samples. The BMNPs exhibited high capture efficiency to E. coli. Transmission electron microscope (TEM) images showed that the BMNPs were bound to the surface of entire E. coli cells. The target bacteria became magnetic so that could be isolated easily from the sample solution by employing an external magnetic field. The concentration of E. coli captured by the BMNPs was then detected by an ATP bioluminescence method. The optimization of ATP measurement was carried out to improve the detection sensitivity. The proposed method was applied to detect the E. coli inoculated into pasteurized milk with low detection limit (20 cfu/mL) and short detection time (about 1 h).     

水溶性CdSe量子点的合成及其作为荧光探针对大肠杆菌的快速检测

采用水相法以谷胱甘肽为稳定剂合成高稳定性的CdSe量子点,利用化学偶联剂的作用使得量子点表面基团与菌体之间的成功结合,对偶联的条件进行了优化.基于荧光分析法建立了一种快速简便的大肠杆菌检测定量分析方法.研究结果表明:合成的量子点具有稳定、荧光性能良好等突出优点.通过偶联剂量子点能与大肠杆菌结合,其荧光强度与大肠杆菌浓度...     

Lysis of Escherichia coli cells by lysozyme: Discrimination between adsorption and enzyme action

The key factors of enzymatic lysis of cells are the interaction between the enzyme and the cell - catalytic and non-catalytic adsorption of enzyme on cell surface. Here, the studies of lysis of intact Escherichia coli cells by chicken egg white lysozyme were performed. It was found that the ionic strength has a dual effect onto the system. On the one hand, the desorption constant of the enzyme increases with the increase of the solution ionic strength, which results in a better enzyme performance. On the other hand, due to the higher osmosis, the cell lysis rate decreases with the increasing of ionic strength of the system. It was found that pH 8.6 and 30 mM NaCl are optimal conditions for lysis of E. coli cells by lysozyme.     

Fluorescence detection of Escherichia coli on mannose modified ZnTe quantum dots

Rapid detection and identification of Escherichia coli(E.coli) is essential to prevent its quickly spread.In this study,a novel fluorescence probe based on ZnTe quantum dots(QDs) modified by mannose(MAN)had been prepared for the determination of E.coli.The results showed that the obtained QDs showed excellent selectivity toward E.coli,and presented a good linearity in range of 1.0×10⁵～1.0×10⁸ CFU/mL.The optimum fluorescence intensity for detecting E. coli was found to be at...     

Inhibitory study of some novel Schiff base derivatives on Staphylococcus aureus by microcalorimetry

The effect of a series of novel Schiff base compounds on Staphylococcus aureus was studied by microcalorimetric method at 37 °C The results showed that all of the organic compounds had the capacity to inhibit the growth of S. aureus in different extent. And the extent and duration of the inhibitory effect on the growth of S. aureus, judged from the rate constant (k), varied with the different structure of the Schiff base compounds. According to the power-time curves, the multiplication rate constant and inhibition ratio were calculated. The growth rate constant of S. aureus (in log phase) in the presence of Schiff base compounds decreased with the increasing of the concentrations of these compounds regularly. The experimental results revealed that the hydrophilicity of Schiff bases had a great influence on their antibacterial activity. Of these Schiff bases, the greater their hydrophilicity, the higher their antibacterial activity. The antibacterial structure-activity relationship (SAR) of Schiff base derivatives was also briefly discussed.     

Fluorescence detection of adenosine-5′-triphosphate and alkaline phosphatase based on the generation of CdS quantum dots

We have developed an analytical method to detect adenosine-5′-triphosphate (ATP) and alkaline phosphatase (ALP) based on the generation of CdS quantum dots (QDs). We demonstrated that Cd²⁺ cation reacts with S²⁻ anion to generate fluorescent CdS QDs in the presence of some certain amount of ATP. With increase in the ATP concentration, the fluorescence intensity of CdS QDs was also enhanced. ATP can be converted into adenosine by the dephosphorylation of ALP, so that the generation of CdS QDs would be inhibited in the presence of ALP. Therefore, this novel analysis system could be applied to assay ATP and ALP based on the growth of fluorescent CdS QDs.     

基于CdSe/ZnS量子点构建多巴胺荧光检测方法的研究

以CdSe/ZnS量子点为荧光探针,基于多巴胺对CdSe/ZnS量子点的荧光猝灭效应,建立了一种可快速测定多巴胺的荧光检测方法。在最优实验条件下(pH7.4,反应时间20min),多巴胺浓度在0.01~0.7μmol/L范围内与CdSe/ZnS量子点的荧光猝灭强度比值呈良好的线性关系(r=0.996),方法检出限为1.6×10-4μmol/L,相对标准偏差为1.2%。与文献报道的方法相比,该方法的检出限更低,更为灵敏,可用于多巴胺注射液及人体尿样中多巴胺的快速检测。     

Synthesis of (±)-5′-methoxyhydnocarpin-D, an inhibitor of the Staphylococcus aureus multidrug resistance pump

The total synthesis of regioisomerically pure (±)-5′-methoxyhydnocarpin-D (6) from commercially available vanillin (7), methyl gallate (9) and 2′,4′,6′-trihydroxyaceophenone (10) is achieved.     

Characterizing mixed phosphonic acid ligand capping on CdSe/ZnS quantum dots using ligand exchange and NMR spectroscopy

The ligand capping of phosphonic acid functionalized CdSe/ZnS core–shell quantum dots (QDs) was investigated with a combination of solution and solid‐state ³¹P nuclear magnetic resonance (NMR) spectroscopy. Two phosphonic acid ligands were used in the synthesis of the QDs, tetradecylphosphonic acid and ethylphosphonic acid. Both alkyl phosphonic acids showed broad liquid and solid‐state ³¹P NMR resonances for the bound ligands, indicative of heterogeneous binding to the QD surface. In order to quantify the two ligand populations on the surface, ligand exchange facilitated by phenylphosphonic acid resulted in the displacement of the ethylphosphonic acid and tetradecylphosphonic acid and allowed for quantification of the free ligands using ³¹P liquid‐state NMR. After washing away the free ligand, two broad resonances were observed in the liquids' ³¹P NMR corresponding to the alkyl and aromatic phosphonic acids. The washed samples were analyzed via solid‐state ³¹P NMR, which confirmed the ligand populations on the surface following the ligand exchange process. Copyright © 2015 John Wiley & Sons, Ltd.     

Aqueous synthesis of CdSe and CdSe/CdS quantum dots with controllable introduction of Se and S sources

A new and convenient route is developed to synthesize CdSe and core-shell CdSe/CdS quantum dots(QDs) in aqueous solution.The gaseous precursors,H2Se and H2S,generated on-line by reducing SeO 3 2à with NaBH 4 and the reaction between Na 2 S and diluted H2SO 4,are used to form high-quality CdSe and CdSe/CdS QDs,respectively.The synthesized water-soluble CdSe and CdSe/CdS QDs possess high quantum yield(3% and 20%) and narrow full-width-at-half-maximum(43 nm and 38 nm).The synthesis process is easily reproducible with simple apparatus and low-toxic chemicals,and can be readily extended to the large-scale aqueous synthesis of QDs.     

Synthesis of magnetic framework composites for the discrimination of Escherichia coli at the strain level

Rapid and efficient characterization and identification of pathogens at the strain level is of key importance for epidemiologic investigations, which still remains a challenge. In this work, solvothermically Fe₃O₄-COOH@MIL-101 composites were fabricated by in situ crystallization approach. The composites combine the excellent properties of both chromium (III) terephthalate (MIL-101) and carboxylic-functionalized magnetite (Fe₃O₄-COOH) particles and possess the efficient peptides/proteins enrichment properties and magnetic responsiveness. Fe₃O₄-COOH@MIL-101 composites as magnetic solid phase extraction materials were used to increase the discriminatory power of MALDI-TOF MS profiles. BSA tryptic peptides at a low concentration of 0.25 fmol μL⁻¹ could be detected by MALDI-TOF MS. In addition, Fe₃O₄-COOH@MIL-101 composites were successfully applied in the selective enrichment of the protein biomarkers from bacterial cell lysates and discrimination of Escherichia coli at the strain level. This work provides the possibility for wide applications of magnetic MOFs to discriminate pathogens below the species level.     

Preparation of highly deuterated zeaxanthin, lycopene, and β-carotene from fully deuterated mevalonate using engineered Escherichia coli

Carotenoids are important natural pigments produced by various microorganisms and plants. Specific deuterium-labeling of these compounds is invaluable in biochemical and physiochemical research. In this paper, preparation of highly deuterated zeaxanthin, lycopene, and β-carotene using engineered Escherichia coli with fully deuterated mevalonate is described. Also described are physico-chemical properties of the obtained deuterated carotenoids.